Differential association of receptor-Gβγ complexes with β-arrestin2 determines recycling bias and potential for tolerance of δ opioid receptor agonists

Abstract

Opioid tendency to generate analgesic tolerance has been previously linked to biased internalization. Here, we assessed an alternative possibility; whether tolerance of delta opioid receptor agonists (DORs) could be related to agonist-specific recycling. A first series of experiments revealed that DOR internalization by DPDPE and SNC-80 was similar, but only DPDPE induced recycling. We then established that the non-recycling agonist SNC-80 generated acute analgesic tolerance that was absent in mice treated with DPDPE. Furthermore, both agonists stabilized different conformations, whose distinct interaction with Gβγ subunits led to different modalities of β-arrestin2 (βarr2) recruitment. In particular, bioluminescence resonance energy transfer (BRET) assays revealed that sustained activation by SNC-80 drew the receptor C terminus in close proximity of the N-terminal domain of Gγ2, causing βarr2 to interact with receptors and Gβγ subunits. DPDPE moved the receptor C-tail away from the Gβγ dimer, resulting in βarr2 recruitment to the receptor but not in the vicinity of Gγ2. These differences were associated with stable DOR-βarr2 association, poor recycling, and marked desensitization following exposure to SNC-80, while DPDPE promoted transient receptor interaction with βarr2 and effective recycling, which conferred protection from desensitization. Together, these data indicate that DORs may adopt ligand-specific conformations whose distinct recycling properties determine the extent of desensitization and are predictive of analgesic tolerance. Based on these findings, we propose that the development of functionally selective DOR ligands that favor recycling could constitute a valid strategy for the production of longer acting opioid analgesics.

Figure 1.

Internalization induced by DPDPE and SNC-80 is similar. A, Cortical neuron cultures were incubated with primary antibody to label surface Flag-DORs. Neurons were then treated with DPDPE or SNC-80 (10 μm) for the indicated times. After stopping treatment, antibody bound to surface receptors was stripped, cells were fixed, permeabilized, and labeled with secondary antibody for visualization of intracellular labeling. A representative example from three independent experiments is shown. B, Histograms correspond to mean intracellular fluorescence intensity quantified in neurons that were exposed to agonists or vehicle for 1 h and represent mean ± SEM of four independent experiments. Internalization data were analyzed together with recycling results shown in Figure 2B by means of repeated measures two-way ANOVA (interaction F_(2,15) = 13.02, p < 0.001). Bonferroni post hoc for multiple comparisons among internalization groups: ***p < 0.001 comparing drugs to vehicle; p > 0.05 comparison among drugs. C, HEK293 cells expressing Flag-DORs were exposed to DPDPE or SNC-80 (1 μm) for the indicated time periods. Receptors at the cell surface were measured by FACs. Results correspond to loss of surface receptors and are expressed as a percentage of receptors present at the membrane before internalization. Data represent mean ± SEM of four independent experiments that were analyzed by repeated measures two-way ANOVA (interaction F_(2,15) = 5.33, p < 0.05). Bonferroni post hoc for multiple comparisons: *p < 0.05 comparing DPDPE to SNC-80 at 15 min. D, HEK293 cells expressing Flag-DORs were exposed to DPDPE or SNC-80 (1 μm) for the indicated time periods. Receptors at the cell surface were measured by an ELISA-based method, as described in the experimental section. Results correspond to loss of surface receptors and are expressed as a percentage of receptors present at the membrane before internalization. Data represent mean ± SEM of six independent experiments that were analyzed by repeated measures two-way ANOVA (interaction F_(5,55) = 0.8, p > 0.05; difference among drugs F_(1,55) = 0.2, p > 0.05).

Figure 2.

Recycling of neuronal DORs is different following exposure to DPDPE and SNC-80. A, Neurons were treated as in Figure 1A following which they were washed and either immediately processed for intracellular labeling (stimulated) or were first allowed to recover for 60 min in the absence of ligand (stimulated + recovery). Labeling of intracellular DORs was done as in Figure 1A. The image shows a representative example of one of four independent experiments. B, Histograms represent mean intracellular intensity in neurons that were stimulated and immediately labeled (dashed), or in neurons that were first allowed to recover in the absence of ligand (filled). Note: dashed bars correspond to internalization values in Figure 1B. Results represent mean ± SEM of four independent experiments. Recycling data were analyzed together with internalization results shown in Figure 1B by means of repeated measures two-way ANOVA (interaction F_(2,15) = 13.02, p < 0.001). Bonferroni post hoc for multiple comparisons: ***p < 0.001 comparing recovery by DPDPE and SNC-80; ^#p < 0.001 comparing stimulated versus stimulated + recovery for DPDPE. C, Neurons were treated and washed as indicated above before allowing them to recover in the absence of ligand. At the end of the recovery period, cells were processed for surface labeling as described in the experimental section. The image shows a representative example of surface labeling obtained in different treatment conditions and corresponds to one example of four independent experiments. D, Histograms represent “recovery ratios” obtained following recovery from exposure to DPDPE or SNC-80 and correspond to mean ± SEM of four independent experiments. “Recovery ratios” were calculated by dividing the number of labeled neurons present in agonist-treated slides by the total number of surface-labeled neurons present in slides that were not subject to treatment. Recovery ratios were analyzed by paired t test; **p < 0.01.

Figure 3.

DPDPE and SNC-80 elicit different DOR recycling in HEK293 cells. A, Cells stably expressing wild-type DORs were incubated with SNC-80 or DPDPE (1 μm; 30 min) to induce internalization. At the end of treatment, cells were washed to remove agonist and allowed to recover for the indicated periods of time before membrane receptors were assessed using an ELISA-based method. Results were expressed as percentage of internalized DORs and represent mean ± SEM of 4–8 independent experiments. Curves were compared by mixed two-way ANOVA (interaction F_(4,50) = 23.8, p > 0.001; difference among drugs p < 0.001). B, HEK293 cells were treated and washed as above before being used in [³H]naltrindole displacement assays in which the radioligand (1.5 nm) was displaced with the indicated concentrations of cold naltrindole. K_d values were calculated from concentrations that inhibited [³H]naltrindole binding by 50% (IC₅₀) using the Cheng–Prusoff equation. K_d CTLs: 223 ± 40 pm; K_d following SNC-80: 302 ± 42 pm; K_d following DPDPE: 280 ± 40 pm.

Figure 4.

DPDPE and SNC-80 differ in their ability to induce desensitization and acute analgesic tolerance. HEK293 cells were incubated with 10 μm cycloheximide and treated or not with monensin (50 μm) before exposing them to vehicle (DMSO 0.01%), DPDPE, or SNC-80 (1 μm; 60 min). At the end of treatment, cells were washed and either immediately used to monitor cAMP accumulation after agonist-induced desensitization (A) or were first allowed to recover in the absence of ligand so as to evaluate resensitization (B). Results are expressed as percentage of maximal inhibition obtained in corresponding untreated controls, and correspond to 5–6 independent experiments performed in duplicates. A, Desensitization data were analyzed by means of three-way ANOVA (agonist × monensin × concentration). Interaction for agonist × monensin groups (F_(2,188) = 7.20, p < 0.01). Bonferroni post hoc for multiple comparisons: control versus desensitization by DPDPE p < 0.001; control versus desensitization by SNC-80 p < 0.001; desensitization by DPDPE versus desensitization by SNC-80 p < 0.001; desensitization by DPDPE versus desensitization by DPDPE + monensin p < 0.001; desensitization by SNC-80 versus desensitization by SNC-80 + monensin p > 0.05. B, Resensitization data were analyzed as above. Interaction for agonist × monensin groups was nonsignificant (F_(2,235) = 1.7, p > 0.05). Effect of agonist factor: F_(2,235) = 267, p < 0.001; effect of monensin factor: F_(1,235) = 0.75, p > 0.05. Bonferroni for post hoc comparisons showed resensitization following DPDPE versus resensitization following SNC-80, p < 0.001. C, One month following induction of the spared nerve injury model of neuropathic pain, calibrated von Frey filaments were used to evaluate the reversal of mechanical hypersensitivity following administration of an intrathecal injection of either vehicle, DPDPE or SNC-80. The 50% withdrawal thresholds (g) were then evaluated three times every 20 min, before administering a second identical dose of the corresponding treatment followed by similar assessment. Results are expressed as mean ± SEM, n = 10–20/treatment group. Data were analyzed using three-way ANCOVA (treatment × testing over time × injection) using basal thresholds (t = 0) as coregressor (F_(1,231) = 22.0, p < 0.001; heterogeneity of regression slope: F_(17,214) = 1.15, p > 0.05). Treatment × injection interaction (F_(1,231) = 5.3, p < 0.05) allowed the following comparisons: effect of first DPDPE injection versus effect of first SNC-80 injection: p > 0.05; effect of second DPDPE injection versus effect of second SNC-80 injection: p < 0.01; effect of first versus second DPDPE injection: p > 0.05; effect of first versus second SNC-80 injection: p < 0.05.

Figure 5.

Sustained exposure to DPDPE and SNC-80 stabilized DORs into conformations that distinctively interact with Gβγ subunits. HEK293 cells were transfected with Gαi-Luc/DOR-GFP (A, B) or DOR-Luc/GFP-Gγ2 (C, D) plus indicated accessory proteins. On the day of the experiment they were exposed to DPDPE (1 μm), SNC-80 (1 μm), or vehicle (0.01% DMSO) for 60 min. At the end of treatment, cells were resuspended, washed, and transferred to a microplate where coelenterazine was added 2 min before BRET2 measures. Results were expressed as the difference between netBRET values obtained in vehicle and agonist-treated cells, and correspond to mean ± SEM of 4–5 independent experiments (A, C). In experiments assessing the acute effect of naltrindole (1 μm) in cells that had been preexposed to DPDPE or SNC-80, the antagonist was introduced 2 min before coelenterazine. Results were expressed as the difference between netBRET values obtained in presence or absence of naltrindole (B, D). Statistical comparisons were done by repeated measures two-way ANOVA on netBRET values. (A, B): agonist × naltrindole interaction was not significant (F_(2,20) = 1.9, p > 0.05); effect of agonist factor (F_(2,20) = 32.9, p < 0.001), effect of naltrindole factor (F_(1,20) = 0.6, p > 0.05); Bonferroni for post hoc comparisons showed ***p < 0.001 comparing drugs to vehicle. (C, D): agonist × naltrindole interaction (F_(2,15) = 9.3, p < 0.01), Bonferroni for post hoc comparisons showed *p < 0.05; ***p < 0.001 comparing agonist to vehicle; ^#p < 0.01 comparing DPDPE versus SNC-80 in CTL groups and in naltrindole groups; ^&p < 0.01 comparing netBRET values in presence and in absence of naltrindole.

Figure 6.

Sustained exposure to DPDPE and SNC-80 stabilized DORs into conformations that were distinctively recognized by [³H]naltrindole. A, HEK293 cells stably expressing Flag-DORs were exposed to DPDPE, SNC-80 (1 μm; 1 h), or vehicle (0.01% DMSO). Following treatment, cells were washed and [³H]naltrindole binding was then assessed. Histograms represent mean ± SEM of B_max binding obtained in four independent experiments. Statistical comparisons were done by repeated measures one-way ANOVA (F_(2,6) = 20.9, p < 0.01) using Bonferroni for post hoc comparisons: *p < 0.05 comparing SNC-80 vs DPDPE; **p < 0.01 comparing control versus DPDPE. Right panel corresponds to a representative example of four independent saturation experiments. B, HEK293 cells expressing Flag-DORs were exposed to agonist or vehicle as above. Following treatment cells were washed and membrane lysates prepared. The solubilization product was then separated by electrophoresis SDS-PAGE and receptor protein was revealed by immunoblot. A representative example of four independent experiments is shown.

Figure 7.

DORs stabilized by DPDPE and SNC-80 distinctively interact with βarr2. HEK293 cells were transfected with recombinant plasmids for the BRET pairs and accessory proteins as indicated in the figure to assess βarr2 recruitment from the vantage point or the receptor (A, B) or the Gβγ dimer (C, D). A, On the day of experiment, HEK293cells were suspended in PBS and distributed into a microplate following which they were introduced into the plate reader and incubated with coelanterazine for 5 min before adding DPDPE or SNC-80 (1 μm). BRET1 measures were obtained every 18 s following agonist addition. Data correspond to one of three independent experiments. B, HEK293 cells were incubated with DPDPE or SNC-80 (1 μm; 37°C) before resuspending them in PBS and taking BRET1 readings 5 min after manual addition of coelenterazine h. Results were expressed as the difference between netBRET values obtained in vehicle and agonist-treated cells and correspond to mean ± SEM of at least five independent experiments. NetBRET values obtained in vehicle-treated cells: 0.021 ± 0.004. Statistical comparisons were done by repeated measures one-way ANOVA on netBRET values. (F_(4,16) = 262.3, p < 0.001) using Bonferroni for post hoc comparisons: ***p < 0.001 comparing drugs to vehicle; ^#p < 0.001 comparing DPDPE versus SNC-80. C, Cells were processed as in A and BRET measures similarly taken. D, Treatment, BRET measures, expression of results, and statistical analyses were performed as in B. Results correspond to mean ± SEM of five independent. netBRET values in vehicle-treated cells: 0.019 ± 0.002. Repeated measures one-way ANOVA (F_(4,16) = 23.6, p < 0.001), using Bonferroni for post hoc comparisons: ***p < 0.001 comparing SNC-80 to vehicle; ^#p < 0.001 comparing DPDPE versus SNC-80.

Figure 8.

The duration of βarr2 association with DOR-Gβγ complexes depends on the activating agonist. HEK293 cells were transfected with recombinant plasmids for the indicated BRET pairs and accessory proteins. On the day of the experiment, they were exposed to 1 μm DPDPE (A, C, E) or SNC-80 (B, D, F) for 30 min. Treatment was stopped and cells washed by addition of PBS, following which they were either immediately used for BRET measures or were first allowed to recover in presence or absence of naltrindole (1 μm) for the indicated periods of time. BRET1 readings were taken 5 min after manual addition of coelenterazine. All data are expressed as percentage of mean netBRET values observed in vehicle-treated controls, and correspond to mean ± SEM of three to six independent experiments. Statistical comparisons for each BRET pair were done on netBRET values using repeated measures one-way ANOVA. A, B: (F_(14,56) = 36.7, p < 0.001), ^#p < 0.001; ^&p < 0.001; ***p < 0.001 comparing treatments to vehicle. C, D: (F_(14,70) = 20.5, p < 0.001); ^&p < 0.001; ***p < 0.001 comparing treatments to vehicle. E, F: (F_(10,20) = 33.9, p < 0.001); ^&p < 0.001; *p < 0.05, ***p < 0.001 comparing treatments to vehicle.

Figure 9.

The stability of βarr2 association to DOR-Gβγ complexes depends on the activating agonist. HEK293 cells expressing or not Flag-DORs, βarr2-Luc, YFP-Gγ2, and accessory heterotrimeric Gαi1 and Gβ1 subunits were treated with 1 μm SNC-80 (A, B) or DPDPE (C, D) for 30 min. At the end of treatment, cells were washed and immediately used in coimmunopurification assays or were first allowed to recover for 30 min in the absence of agonist. Cells were then lysed, Flag-DORs purified, and the resulting product separated by SDS-PAGE. Blots show representative examples of the amounts of βarr2-Luc and YFP-Gγ2 that were copurified with Flag-DORs in the following: (1) non-transfected HEK293 cells; (2) transfected cells that were not exposed to agonist; (3) transfected cells that were exposed to agonist during 30 min, and (4) transfected cells that were exposed to agonist and then allowed to recover for 30 min in the absence of ligand. Insets show control blots of proteins amounts present in lysates before immunopurification and amount of Flag-DOR immunopurified for each sample. Histograms show results obtained by compiling densitometric measures of eight independent experiments. Data are expressed as percentage of βarr2-Luc/Flag-DOR or YFP-Gγ2/Flag-DOR ratios in controls. Statistical comparisons of immunoreactivity ratios were done by repeated measures one-way ANOVA. βarr2-Luc association with Flag-DORs (A, C): (F_(4,28) = 10.0, p < 0.001); *p < 0.05, **p < 0.01 comparing treatments to vehicle; ^#p < 0.05 comparing βarr2-Luc/Flag-DORs ratios following recovery or not from DPDPE treatment. YFP-Gγ2 association with Flag-DORs (B, D): (F_(4,28) = 13.8, p < 0.001); *p < 0.05; **p < 0.01 comparing treatments to vehicle.

Figure 10.

Naltrindole allows us to rescue DOR recycling following SNC-80 exposure. HEK293 cells stably expressing wild-type DORs were incubated with SNC-80 (A) or DPDPE (B; 1 μm, 30 min) to induce internalization. At the end of treatment, cells were washed to remove agonist and allowed to recover for the indicated periods of time in the presence or absence of the indicated ligands. At the end of each recovery period, membrane receptors were assessed using an ELISA-based method. Receptors recovered at the membrane were expressed as percentage of internalized DORs and represent mean ± SEM of 4–9 independent experiments. Data were analyzed with mixed two-way ANOVA. A, Time × treatment interaction for SNC-80 (F_(15,156) = 4.3, p < 0.001); using Bonferroni for post hoc comparisons: p < 0.001 comparing SNC-80 to SNC-80 + naloxone or to SNC-80 + naltrindole; p > 0.05 comparing SNC-80 to SNC-80 + morphine. B, No time × treatment interaction was observed for DPDPE (F_(5,48) = 0.4, p > 0.05), p > 0.05 comparing DPDPE to DPDPE + naltrindole.